Urolithin A attenuates auditory cell senescence by activating mitophagy

Aging of sensory organs is associated with a decline in mitochondrial function and the accumulation of dysfunctional mitochondria. Impaired mitophagy blocks the turnover of dysfunctional mitochondria and leads to their accumulation. Urolithin A (UA) induces mitophagy in various mammalian cells. This study was aimed at investigating the effect of the mitophagy activator, UA, on premature senescent auditory cells. The levels of cellular senescence-associated p53 and p21 significantly increased in H2O2-induced senescent House Ear Institute‐Organ of Corti 1 (HEI-OC1) cells and cochlear explants. However, the levels of mitophagy-related molecules significantly decreased. UA significantly decreased the expression of senescence-associated p53 and p21, and increased the expression of mitophagy-related proteins, in H2O2-induced senescent cells and cochlear explants. The percentage of β-galactosidase-stained senescent cells also reduced in H2O2-treated cells and cochlear explants upon UA pre-treatment. The formation of mitophagosomes and mitophagolysosomes was restored upon UA pre-treatment of H2O2-induced senescent cells. The knockdown of mitophagy-related genes (Parkin and Bnip3) resulted in annulment of UA-induced anti-senescent activity. UA significantly increased the ATP content, mitochondrial DNA (mtDNA) integrity, and mitochondrial membrane potential in senescent HEI-OC1 cells. These findings indicate that UA counteracted mitophagy decline and prevented premature senescence in auditory cells. Hence, UA administration might be a promising strategy for preventing mitochondrial dysfunction in patients with age-related hearing loss.

tigate premature cellular senescence and the changes in mitophagy-related proteins upon the application of H 2 O 2 to HEI-OC1 cells and cochlear explants, senescence-associated p53 and p21, and mitophagy-related proteins, were examined. The levels of cellular senescence-associated p53 and p21 significantly increased in H 2 O 2 -treated cells and cochlear explants. However, the levels of mitophagy-related proteins-PINK1, Parkin, BNIP3, and LC3B-significantly decreased (Fig. 1A,B). Cellular senescence was examined using β-galactosidase activity; senescence was induced in HEI-OC1 cells 5 days after H 2 O 2 treatment at a rate of 44.9% ± 2.1% (Fig. 1C,D). The population doubling rate indicates the speed of cell growth. The population doubling rate of H 2 O 2 -treated cells decreased (2.41 ± 0.1) compared with that of normal cells (4.57 ± 0.1; Fig. 1E). These results demonstrated that H 2 O 2 treatment induced cellular senescence and reduced the levels of mitophagy-related proteins in auditory cells.
UA counteracts mitophagy decline and decreases cellular senescence. To examine if UA is toxic to cells, cell viability was assessed after UA treatment. UA did not decrease the cell viability of HEI-OC1 cells ( Fig. 2A). To examine the effect of UA on mitophagy and cellular senescence, senescence-associated markers and mitophagy-related genes-Pink1, Parkin, and Bnip3-in H 2 O 2 -induced senescent HEI-OC1 cells were examined. The gene (mRNA) expression of senescence markers was determined using the senescence-associated secretory phenotype and a classical hallmark of cellular senescence, CDKN1a (p21). Significant increases in the mRNA expression of the senescence markers, CDKN1a (p21), IL1a, Cxcl2, IL6, Timp1, Ccl20, and Actb, were detected in the H 2 O 2 -treated group. The expression of mitophagy-related genes significantly decreased in the H 2 O 2 -treated group. UA pre-treatment before H 2 O 2 treatment significantly increased the mRNA levels of mitophagy-related genes and decreased the mRNA levels of senescence markers, compared with those in cells treated with H 2 O 2 alone (Fig. 2B,C). UA also significantly decreased the expression of senescence-associated p53 and p21, whereas it significantly increased the expression of mitophagy-related proteins in H 2 O 2 -induced senescent HEI-OC1 cells (Fig. 3A). The formation of mitophagosomes was investigated using immunofluorescence analysis of the co-localization of LC3B and MitoTracker. This co-localization significantly decreased in H 2 O 2 -treated cells, but significantly increased upon UA pre-treatment (Fig. 3B, upper panel). The formation of mitophagolysosomes was investigated using immunofluorescence analysis of the co-localization of LAMP1, a marker of lysosomes, and MitoTracker. This co-localization significantly decreased in H 2 O 2 -treated cells, but increased upon UA pre-treatment (Fig. 3B, lower panel). The percentage of β-galactosidase-stained senescent cells reduced in H 2 O 2 -treated cells upon UA pre-treatment (Fig. 3C). UA significantly decreased the expression of senescence-associated p53 and p21, whereas it significantly increased the expression of mitophagy-related proteins, in H 2 O 2 -treated cochlear explants (Fig. 4A). The percentage of β-galactosidase-stained senescent cells reduced in H 2 O 2 -treated cochlear explants upon UA pre-treatment (Fig. 4B). To investigate whether antisenescent activity of UA is dependent on mitophagy or by other effects, knockdown of mitophagy genes was conducted in HEI-OC1 cells. Knockdown of Parkin and Bnip3 mRNAs significantly reduced the formation of mitophagosomes and mitophagolysosomes (Fig. 5A). While the percentage of β-galactosidase-stained senescent cells reduced in si-Control H 2 O 2 -treated cells upon UA pre-treatment, the percentage of β-galactosidase-stained senescent cells did not reduce post siRNA-mediated knockdown of Parkin and Bnip3 (Fig. 5B). These results indicated that UA induced mitophagy and attenuated premature senescence in auditory cells.
UA increases mitochondrial function in senescent HEI-OC1 cells. ATP content, mtDNA integrity, and mitochondrial depolarization were examined in H 2 O 2 -induced senescent HEI-OC1 cells to investigate the beneficial effect of UA on mitochondrial function. The ATP content in the H 2 O 2 -treated group was significantly lower than that in the control group; however, pre-treatment with UA significantly increased the ATP content following H 2 O 2 treatment (Fig. 6A). mtDNA integrity was assessed as the ratio of long mtDNA to short mtDNA. The mtDNA integrity of the H 2 O 2 treatment group was significantly lower than that of the control group; however, the mtDNA integrity of the UA pre-treatment before H 2 O 2 treatment group was significantly higher than that of the H 2 O 2 treatment group (Fig. 6B). Mitochondrial membrane potential is a parameter necessary for functional mitochondria. The mitochondrial depolarization values of the H 2 O 2 treatment group were significantly higher than those of the control group. However, UA pre-treatment significantly decreased the mitochondrial depolarization values following H 2 O 2 treatment (Fig. 6C). These results indicated that UA increased the mitochondrial integrity and function in senescent auditory cells.

Discussion
Mitochondria generate ATP in cells and are important for energy supply. They are also critical for neurotransmission in the auditory system 15 . The numbers of damaged and dysfunctional mitochondria increase with age, triggering the production of reactive oxygen species and the consequent age-dependent decline in organ function 16,17 . Mitophagy maintains a healthy mitochondrial population through the efficient removal of damaged mitochondria, and contributes to mitochondrial quality control 18 .
In this study, oxidative stress-induced premature senescence was associated with a progressive decrease in the expression of mitophagy-related genes and proteins. The PINK1/Parkin pathway is a major pathway of mitophagy for mitochondrial quality control in mammalian cells. PINK1 recruits Parkin upon the loss of mitochondrial membrane potential. This subsequently activates the ubiquitin-proteasome system and promotes the engulfment of damaged mitochondria through the formation of mitophagosomes 19 . BNIP3 is a key factor in Parkin-independent regulation of mitophagy 20 . It binds to LC3 on autophagosomes to promote the autophagic www.nature.com/scientificreports/ engulfment of mitochondria 4 . The decrease in the expression of mitophagy-related genes and proteins (PINK1, Parkin, and BNIP3) in premature senescent cells indicated a decline in mitophagy, and the accumulation of damaged mitochondria. Competent mitochondrial activity is highly associated with mtDNA integrity and ATP production 21 . Through the present study, we have provided evidence that UA restores mitochondrial integrity and function, including ATP production and mitochondrial membrane potential, in senescent auditory cells. Damaged mitochondria are engulfed by autophagosomes, which subsequently fuse with lysosomes and form mitophagolysosomes during HEI-OC1 cells were exposed to 2 mM H 2 O 2 for 1 h and incubated for 3, 4, and 5 days. Whole cochleae also were exposed to 0.5 mM H 2 O 2 for 5 h and incubated for 1, 3, and 5 days. The levels of p53 and p21 (cellular senescence markers) increased in H 2 O 2 -treated HEI-OC1 cells (A) and cochlear explants (B). The levels of mitophagy-related proteins (PINK1, Parkin, BNIP3, and LC3B) significantly decreased in H 2 O 2treated HEI-OC1 cells (A) and cochlear explants (B). HEI-OC1 cells were exposed to 2 mM H 2 O 2 for 1 h and incubated for 5 days. β-galactosidase staining of senescent cells significantly increased in H 2 O 2 -treated cells (C, D). The population doubling level significantly decreased in H 2 O 2 -treated cells (E). Data are presented as means ± standard errors of the mean of five independent experimental results (SA-β-gal, senescence associatedbeta galactosidase). *p < 0.05, **p < 0.01. The grouping of gels/blots cropped from different parts of the same gel. Full-length blots are presented in Fig. S1A, S1B. Mitochondria-targeted therapeutic strategies for pathologies associated with aging have received attention due to the effect of enhancing mitochondrial function 12 . Mitophagy maintains neuronal function through maintaining a healthy mitochondrial population and efficient energy supply in the nervous system 23 . Mitophagy inducers such as Kaempferol and Rhapontigenin inhibited memory loss in nematode and rodent models of Alzheimer's disease through the elimination of dysfunctional mitochondria 24 . Induction of mitophagy through NAD + augmentation forestalled Alzheimer's disease, Parkinson's disease, and Huntington disease in animal and cell culture models 25 . In contrast, inactivation of PINK1 resulted in neuronal degeneration and Huntington disease 26 .
UA has previously been reported to exert anti-aging effects in several cells 27 . UA improves mitochondrial respiratory capacity and induces the expression of mitochondrial oxidative phosphorylation proteins 11 . It also exhibits multiple biological activities, including antioxidant, anti-inflammatory, anticancer, and antimicrobial properties 28,29 . The biological effects of UA in neurodegeneration and diseases of the central nervous system have been reported 30 . UA improved associative memory and neuronal survival through the induction of PINK1 expression in neurons of an Alzheimer's disease mouse model 31 . UA also protected against ischemic stroke induced by occlusion of the cerebral artery 32 . In addition, UA exhibited neuroprotective effects in a mouse model of multiple sclerosis by reducing white matter demyelination 33 .
It has been reported that UA supplementation in humans is suitable, in terms of safety and bioavailability, to improve mitochondrial and cellular health. Nutritional intervention with UA can be applied to humans for promoting healthy mitochondrial function throughout their lifetimes 12 .
Our results demonstrated that UA counteracted mitophagy decline and maintained mitochondrial function in premature senescent auditory cells. UA administration might be a promising strategy for preventing a decline in mitochondrial function in auditory cells, and age-related hearing loss.

Conclusion
This study proved that UA induces mitophagy and prevents premature senescence in auditory cells. The activation of mitophagy using UA can be a potential preventive strategy for patients with age-related hearing loss.   All experiments were performed in accordance with the ARRIVE guidelines and strict regulations approved by the committee. The mice were euthanized by cervical dislocation under 5% isoflurane anesthesia. Cochleae and organ of Corti explants were isolated from postnatal day 4 mice for further studies. Each organ of Corti explant was pre-treated with 30 µM UA for 2 h, exposed to 0.5 mM H 2 O 2 for 5 h, replaced in fresh culture medium, and incubated for 1, 3, and 5 days.
Senescence-associated β-galactosidase activity assay. Senescence-associated β-galactosidase activity was measured using the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, MA, USA), according to the manufacturer's protocol. Briefly, cultured and treated cells in a 6-well plate were washed twice with PBS and fixed in 4% paraformaldehyde in PBS for 15 min. After washing twice with PBS, 1 mL staining solution was added to each well, and the plate was sealed with parafilm, incubated at 37 °C overnight (no CO 2 ), and imaged using an ECLIPSE Ti2-E microscope (Nikon, Tokyo, Japan). Cultured and treated cochlear explants